Mechanisms of release and signaling functions of ciliary vesicles

睫状囊泡的释放机制和信号功能

• 批准号: RGPIN-2022-04551
• 负责人: Belleannee, Clemence
• 金额: $ 2.91万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760653
• 关键词: Mechanisms; release; signaling; functions; ciliary

In the mammalian male reproductive system, extracellular vesicles released by somatic epithelial cells participate to the maturation of spermatozoa through the transfer of molecular cargos. This unique process of soma-to-sperm communication occurs in the tubule attached to the testis, named the epididymis, and supports the acquisition of sperm motility and fertilizing abilities. Despite the important role played by extracellular vesicles to sustain male reproductive functions, their cellular source, mechanisms of release and respective signaling functions remain largely unknown. Primary cilia are solitary sensory antennae that protrude upward from the cell surface to form a subcellular compartment enriched in signaling molecules. Vesicles derived from these organelles have been shown to contain a specific subset of bioactive molecules. We recently unveiled that primary cilia are abundant in the tubules located upstream of the epididymis, i.e. in the efferent ductules. In this location, they release extracellular vesicles that express specific ciliary markers (e.g Arl13b and Cytochrome p450 oxidoreductase (POR)) into the epididymal fluid. With the hypothesis that ciliary extracellular vesicles from the epididymis may constitute news means of communication between ciliated somatic cells and the maturing spermatozoa, we propose a research program articulated around 3 majors aims: Aim 1. Characterize ciliary vesicles from the male reproductive system We will take advantage of a double transgenic mouse model, in which the Arl13b ciliary marker is fused with m-cherry tag, to detect and sort primary cilia-deriving extracellular vesicles by dedicated small particle flow-cytometry approaches (collaboration E. Boilard, U.Laval). The size, protein markers and molecular content of m-cherry positive ciliary vesicles will be determined and compared with m-cherry negative extracellular vesicles from the epididymis. Aim 2. Determine their mechanisms of release Vesicles that derive from the primary cilium of endothelial cells are formed by outward budding of the plasma membrane under the control of shear stress and intraflagellar transport (IFT) components. We will test the contribution of these two factors on epithelial primary cell cultures isolated from the efferent ductules of wild type and IFT88 knock-out mice by using biofludic imaging system. Aim 3. Assess their ability to modulate sperm content and function Since extracellular vesicles participate to intercellular communication via the transfer of bioactive molecules, we will assess their role in somatic cells-spermatozoa communication by combining in vitro co-incubation assays with in vivo functional studies on knock-out mouse models. This NSERC research will unravel the unique features and functions of ciliary vesicles in the soma-to-sperm communication system and may identify new cellular mechanisms that control sperm maturation and male reproductive functions.

在哺乳动物雄性生殖系统中，体细胞上皮细胞释放的细胞外囊泡通过转运分子物质参与精子的成熟。这种独特的体细胞与精子通讯过程发生在连接睾丸的小管（称为附睾）中，并支持精子运动和繁殖能力的获得。尽管细胞外囊泡在维持雄性生殖功能方面发挥着重要作用，但其细胞来源、释放机制和各自的信号功能仍在很大程度上未知。初级纤毛是从细胞表面向上突出以形成富含信号分子的亚细胞区室的孤立感觉触角。来自这些细胞器的囊泡已被证明含有生物活性分子的特定子集。我们最近发现，初级纤毛丰富的小管位于上游的附睾，即在传出小管。在该位置，它们将表达特异性纤毛标记物（例如Arl 13 b和细胞色素p450氧化还原酶（POR））的细胞外囊泡释放到附睾液中。假设从附睾纤毛细胞外囊泡可能构成纤毛体细胞和成熟精子之间的通讯的新手段，我们提出了一个研究计划，围绕3个主要目标：目标1。我们将利用双转基因小鼠模型，其中Ar 113 b纤毛标记物与m-cherry标签融合，通过专用的小颗粒流式细胞术方法检测和分选初级纤毛衍生的细胞外囊泡（协作E. Boilard，U.拉瓦尔）。将测定m-cherry阳性纤毛囊泡的大小、蛋白质标志物和分子含量，并与附睾的m-cherry阴性细胞外囊泡进行比较。 目标2.确定它们的释放机制来自内皮细胞初级纤毛的囊泡是在剪切应力和鞭毛内运输（IFT）成分的控制下通过质膜向外出芽形成的。我们将使用生物流体成像系统测试这两个因素对从野生型和IFFT 88基因敲除小鼠的输出小管分离的上皮原代细胞培养物的贡献。 目标3.评估它们调节精子含量和功能的能力由于细胞外囊泡通过生物活性分子的转移参与细胞间通讯，我们将通过将体外共孵育测定与敲除小鼠模型的体内功能研究相结合来评估它们在体细胞-精子通讯中的作用。这项NSERC研究将揭示纤毛囊泡在胞体-精子通讯系统中的独特特征和功能，并可能确定控制精子成熟和男性生殖功能的新细胞机制。